1. Field of the Invention
The present invention relates to a novel monoclonal antibody, more particularly, to a monoclonal antibody which is specific to a polypeptide capable of inducing the interferon-xcex3 (hereinafter abbreviated as xe2x80x9cIFN-xcex3xe2x80x9d) production by immunocompetent cells.
2. Description of the Prior Art
It is known that IFN-xcex3 is a protein which has antiviral- , antioncotic- and, immunoregulatory-activities, and is produced by immunocompetent cells stimulated with antigens or mitogens. Because of these biological activities, IFN-xcex3 is expected to be used as an antitumor agent from the beginning of the finding, and is studied energetically on clinical trials as a therapeutic agent for malignant tumors in general including brain tumors. IFN-xcex3 preparations now commercially available are roughly classified into 2 groups, i.e. natural IFN-xcex3s produced by immunocompetent cells and recombinant IFN-xcex3s produced by transformants obtained by introducing into microorganisms of the species Escherichia coli DNAs which encode such natural IFN-xcex3s. In the above clinical trials, one of these IFN-xcex3s is administered to patients as an xe2x80x9cexogenous IFN-xcex3xe2x80x9d.
Among these IFN-xcex3s, natural IFN-xcex3s are usually produced by culturing established immunocompetent cells in nutrient culture media supplemented with IFN-xcex3 inducers to form IFN-xcex3s, and purifying the formed IFN-xcex3s. It is known that the type of IFN-xcex3 inducers greatly influence on the IFN-xcex3 yield, as well as the facility of IFN-xcex3 purification and the safety of the final products. Generally, mitogens such as concanavalin A (Con A), Lens culinaris, Phytolacca americana, endotoxin and lipopolysaccharide are used as an IFN-xcex3 inducer. However, these mitogens have problems related to their molecular varieties and quality changes depending related to their origins and purification methods, as well as the difficulty of obtaining a desired amount of preparations with a constant IFN-xcex3 inducibility. In addition, most of these mitogens induce unfavorable side effects when administered to living bodies, and some of them even cause toxicity, so that it is substantially difficult to induce the IFN-xcex3 production by direct administrations to living bodies.
The present inventors found in mouse liver a substance which induces the, IFN-xcex3 production during their research of cytokines produced from mammalian cells. They isolated the substance by using a variety of purification methods comprising column chromatography as a main, technique, and studied the properties and features, revealing, that the substance was a protein having the following physicochemical properties:
(1) Molecular weight Exhibiting a molecular weight of 19,000xc2x15,000 daltons on sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE);
(2) Isoelectric point (pI) Exhibiting an isoelectric point of 4.8xc2x11.0 on chromatofocusing;
(3) Partial amino acid sequence Having the partial amino acid sequences in SEQ ID NOs: 4 and 5; and
(4) Biological activity Inducing the IFN-xcex3 production by immunocompetent cells.
It can be concluded that it is a novel substance because no protein with these physicochemical properties has been known. The present inventors continued studies on mouse liver cells and have found that the DNA SEQ ID NO:6 of the substance consists of 471 base pairs and encodes the amino acid sequence in SEQ ID NO:7.
Based on these findings, the present inventors continued studies on human liver cells and have obtained a DNA which encodes another novel substance that induces IFN-xcex3 production by immunocompetent cells. They revealed that the substance is a polypeptide and decoded its DNA and revealed that the polypeptide has the amino acid sequence in SEQ ID NO:1. They introduced the DNA into Escherichia coil to express the polypeptide and obtained the polypeptide in the resultant culture in a considerably high yield. These findings were disclosed in Japanese Patent Application Nos. 184,162/94 and 304,203/94, applied for by the present inventors.
As is described above, the polypeptide has a property of inducing the IFN-xcex3 production by immunocompetent cells, and is expected to be used in a variety of fields as an IFN-xcex3 inducer, antiviral agent, antitumor agent, antibacterial agent, immunoregulatory agent, and blood platelet enhancing agent. In general, the developments of methods for efficiently purifying biologically active polypeptides to give a relatively-high purity and those for assaying many samples in parallel are inevitably required when the polypeptides should be incorporated into pharmaceuticals. Although the best material enabling these purification and assay is a monoclonal antibody, none of which specific to the polypeptide has been established.
In view of the foregoing, the object of the present invention is to provide a monoclonal antibody which is specific to the polypeptide.
It is another object of the present invention to provide a hybridoma capable of producing the monoclonal antibody.
It is further object of the present invention to provide a method for preparing the monoclonal antibody.
It is yet another object of the present invention to provide a purification method for purifying the polypeptide using the monoclonal antibody.
It is another object of the present invention to provide a detection method for assaying the polypeptide using the monoclonal antibody.
The first object of the present invention is attained by a monoclonal antibody which is specific to a polypeptide having either the amino acid sequence in SEQ ID NO:1 or a homologous amino acid sequence thereunto, and induces IFN-xcex3 production by immunocompetent cells.
The second object of the present invention is attained by a hybridoma capable of producing the monoclonal antibody.
The third object of the present invention is attained by a process for preparing the monoclonal antibody comprising culturing the hybridoma capable of producing the antibody in vitro, i.e. in a nutrient culture medium, or in vivo, i.e. in an animal, and collecting the antibody from the resultant culture or the body fluid.
The fourth object of the present invention is attained by a purification method for polypeptide comprising contacting the monoclonal antibody with a mixture containing the polypeptide and impurities to adsorb the polypeptide thereunto, and desorbing the polypeptide from the antibody.
The fifth object of the present invention is attained by a method for detecting the polypeptide comprising contacting samples with the monoclonal antibody to effect immunological reaction to detect the polypeptide.